Method of inducing a proinflammatory immune response to human α-lactalbumin

ABSTRACT

Compositions and methods for immunization against human breast cancer are disclosed. A breast cancer vaccine comprises an immunogenic polypeptide comprising human α-lactalbumin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/157,990, filed Jun. 10, 2011, and claims the benefit under 35U.S.C. §119(e) of U.S. Provisional Application Ser. No. 61/353,464,filed Jun. 10, 2010, U.S. Provisional Application Ser. No. 61/353,470,filed Jun. 10, 2010, and U.S. Provisional Application Ser. No.61/353,825, filed Jun. 11, 2010, the disclosures of each of which arehereby incorporated herein by reference in their entirety.

GOVERNMENT RIGHTS

This invention was made with government support under grant numbersRO1A1-51837, RO1DC-006422, and RO1CA14035 awarded by the NationalInstitutes of Health. The U.S. Government has certain rights in theinvention.

TECHNICAL FIELD

The present disclosure generally pertains to the fields of immunologyand oncology. More particularly, the present disclosure pertains to theprevention or treatment of breast cancer by vaccination.

BACKGROUND AND SUMMARY

Breast cancer is responsible for the second overall cause ofcancer-related deaths among women. Currently, prevention of breastcancer predominantly involves reducing modifiable risks including earlydetection through physical examination and mammograms, avoidance ofunnecessary post-menopausal hormone therapy, reduction in alcoholconsumption, loss of weight, increase in physical activity, and genetictesting for mutations of the breast cancer type 1 and type 2susceptibility genes (BRCA1 and BRCA2, respectively). More aggressiveapproaches in high risk patients include chemoprevention with tamoxifen,raloxifene, and aromatase inhibitors as well as prophylactic bilateralmastectomy and oophorectomy.

Despite the profound health risk of breast cancer and inadequacy ofpreventative efforts, an immunotherapy for breast cancer has not beendeveloped as an integral part of the standard of care. Tumor-specificantigens have long provided less than optimal results as targets forcancer vaccination. The overall goal of cancer vaccination hastraditionally been to boost the latent immune response to tumor-specificantigens. Approaches have included cell-based protocols involvingimmunization with whole autologous or allogeneic tumors, as well asantigen-based strategies involving immunization with proteins orpeptides overexpressed in tumors and underexpressed in normal tissues.The human epidermal growth factor receptor 2 (HER2) and mucin (MUC1) arethe predominant antigens used in human breast cancer vaccine trials.Although vaccination using these antigens may demonstrate tumor reducingeffects, neither antigen provides any tissue or tumor specificity sinceboth are expressed in a variety of normal tissues and tumors. Thus, thelack of inherent tissue specificity of HER2 and MUC1 targeted immunitymay ultimately lead to substantial systemic autoimmune sequelae if arobust immune response manifests.

A full-strength autoimmune attack sufficient to induce targeted breastfailure can provide effective therapy against established breastmalignancies if the target antigen is constitutively expressed in breasttumors. Moreover, if the selected target antigen is expressed in normalbreast tissue under conditions that are easily avoidable, then thevaccine may provide safe and effective protection against thedevelopment of breast cancer.

Human alpha-lactalbumin (α-lactalbumin) is a conditionally expressed,breast specific differentiation protein found in the majority of breastmalignancies. As an integral differentiation protein involved inregulation of lactose biosynthesis, expression of α-lactalbumin isbreast-specific and conditionally dependent on lactation for itsexpression and synthesis. Human α-lactalbumin is also constitutivelyoverexpressed in the majority of breast tumors, is breast specific, andis sufficiently immunogenic to induce an effective proinflammatoryimmune response. Thus, immunization against human α-lactalbumin offers asafe and effective vaccination strategy for the prevention of breastcancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show the immunogenicity of recombinant mouse α-lactalbumin.Lymph node cells are evaluated 10 days after immunization of SWXJ femalemice with α-lactalbumin and show recall responses that are a)antigen-specific to recombinant mouse α-lactalbumin but not torecombinant human cochlin over a dose range (see FIG. 1A); b) elicitedfrom both purified CD4⁺ and CD8⁺ T cells in response to 25 pg/mlα-lactalbumin (see FIG. 1B); and c) consistent with a proinflammatorytype 1 cytokine profile with high production of IFNγ and IL-2 and lowproduction of the type 2 cytokines, IL-4, IL-5, and lactalbumin (SeeFIG. 1C). All error bars show ±SEM.

FIG. 2 shows the analysis of breast tissue during autoimmune-inducedbreast failure. Real-time RT-PCR analysis of lactating mammary tissueshows significantly elevated expression levels of IFNγ (p=0.001) but notIL-10 (p>0.10). All error bars show ±SEM. Each * indicates astatistically significant difference.

FIGS. 3A and 3B show that α-lactalbumin vaccination prophylacticallyinhibits growth of breast tumors. The growth of autochthonous breasttumors is significantly inhibited in ten month old MMTV-neu miceimmunized with α-lactalbumin at eight weeks of age (p=0.0004; FIG. 3A).Growth of transplanted 4T1 tumors is significantly inhibited followingprophylactic immunization with α-lactalbumin 13 days prior to tumorinoculation (p=0.0006; FIG. 3B). All error bars show ±SEM. Each *indicates a statistically significant difference.

FIGS. 4A-4C show that α-lactalbumin vaccination treats establishedgrowing transplanted breast tumors. Significant inhibition of 4T1 tumorgrowth occurs following α-lactalbumin immunization at 5 days after tumorinoculation (p<0.01; FIG. 4A) and at 13 days after tumor inoculation(p<0.01; FIG. 4B), but not at 21 days after tumor inoculation (p>0.10;FIG. 4C). All error bars show ±SEM. Each * indicates a statisticallysignificant difference.

FIG. 5 shows that α-lactalbumin vaccination treats established growingautochthonous breast tumors. Significant inhibition (p<0.0006) in thegrowth of extremely aggressive autochthonous tumors occurs followingα-lactalbumin immunization of MMTV-PyVT transgenic mice at 6 weeks ofage. Due to massive multifocal tumor growth, tumors in MMTV-PyVT miceare amenable to measurement in only one direction. The longestmeasurements on all ten MMTV-PyVT tumors are added to calculate totaltumor load in mm on each day.

FIGS. 6A-6C show that α-lactalbumin-specific T cells induce tumorinflammation and cytotoxicity. Recall responses to α-lactalbumin asmeasured by ELISA demonstrate a type-1 proinflammatory phenotypeinvolving high production of IFNγ compared to IL-5 and IL-10 (FIG. 6A).ELISPOT analysis of TILs shows that CD4⁺ rather than CD8⁺ T cellsproduce IFNγ (FIG. 6B). Death of cultured 4T1 tumor cells is inhibitedby treatment of cultured α-lactalbumin primed LNC with antibodiesspecific for mouse CD8, indicating that CD8⁺ T cells mediate 4T1specific cytotoxicity (FIG. 6C).

FIGS. 7A-7D show that inhibition of tumor growth by α-lactalbuminvaccination is mediated by T cells. The transfer of α-lactalbumin primedLNC into naïve recipient BALB/c mice on the same day as inoculation with4T1 tumors results in a) a significant inhibition of tumor growth(p<0.0001; FIG. 7A); b) a significant decrease in incidence of tumorbearing mice (p<0.03; FIG. 7B); and c) a significant decrease in finaltumor weight (p<0.0008; FIG. 7C). Compared to ovalbumin (OVA) primedLNC, significant tumor growth inhibition occurs in naïve mice receivingeither CD4⁺ T cells (p=0.002; FIG. 7D left panel) or CD8⁺ T cells(p=0.003; FIG. 7D right panel) that are enriched by magnetic beadseparation from α-lactalbumin primed LNC. All error bars show ±SEM.Each * indicates a statistically significant difference.

FIG. 8 shows in vitro priming of human peripheral blood mononuclearcells (PBMC) T cell using blood derived dendritic cells (DCs) to testfor the availability of a human α-lactalbumin-reactive T cellrepertoire. Priming of PMBCs with α-lactalbumin results in an increasedfrequency of IFNγ producing T cells upon subsequent presentation ofα-lactalbumin (recall response).

DETAILED DESCRIPTION

While the invention is susceptible to various modifications andalternative forms, specific embodiments will herein be described indetail. It should be understood, however, that there is no intent tolimit the invention to the particular forms described, but on thecontrary, the intention is to cover all modifications, equivalents, andalternatives falling within the scope of the invention.

In one embodiment, a human breast cancer vaccine comprising animmunogenic polypeptide is disclosed. The immunogenic polypeptidecomprises human α-lactalbumin according to the amino acid sequence:

(SEQ ID NO: 1) kqftkcelsq llkdidgygg ialpelictm fhtsgydtqaivennestey glfqisnklw ckssqvpqsr nicdiscdkflddditddim cakkildikg idywlahkal ctekleqwlc ekl.

It is appreciated that human α-lactalbumin is processed in vivo byproteases to smaller peptide fragments, which are able to bind to MHCclass I and/or MHC class II molecules on antigen presenting cells.Subsequently, T-cell receptors recognize and bind to the MHC molecule towhich the peptide is bound, forming the primary signal that initiates animmune response.

In one embodiment, the vaccine further comprises an adjuvant and apharmaceutically acceptable carrier. As used herein, the term “adjuvant”refers to an agent that stimulates the immune system and increases theresponse to a vaccine. Vaccine adjuvants are well-known to those ofskill in the art. Illustratively, GPI-0100 is a suitable adjuvant for avaccine. As used herein, the term “carrier” refers to an ingredientother than the active component(s) in a formulation. The choice ofcarrier will to a large extent depend on factors such as the particularmode of administration or application, the effect of the carrier onsolubility and stability, and the nature of the dosage form.Pharmaceutically acceptable carriers for polypeptide antigens are wellknown in the art.

In one embodiment, the vaccine is administered prophylactically toprevent breast cancer. In one illustrative aspect, the vaccine isadministered to non-lactating women at risk for developing breastcancer.

In one embodiment, the vaccine is administered to inhibit tumor cellexpansion. The vaccine may be administered prior to or after thedetection of breast tumor cells in a patient Inhibition of tumor cellexpansion is understood to refer to preventing, stopping, slowing thegrowth, or killing of tumor cells.

In one illustrative aspect, T cells of the human immune system areactivated after administration of an immunogenic composition comprisinghuman α-lactalbumin. The activated T cells may be CD4⁺ and/or CD8⁺.

In one embodiment, after administration of a vaccine comprising humanα-lactalbumin, a proinflammatory response is induced by subsequentencounter of immune cells with α-lactalbumin. The proinflammatory immuneresponse comprises production of proinflammatory cytokines and/orchemokines, for example, interferon gamma (IFNγ) and/or interleukin 2(IL-2). Proinflammatory cytokines and chemokines are well known in theart.

It is to be appreciated that when the breast cancer vaccine isadministered to patients whose breast tissue is not actively producinghuman α-lactalbumin in appreciable quantities (i.e. a non-lactatingfemale, or a female devoid of α-lactalbumin producing breast tumorcells), immunization with human α-lactalbumin does not elicit asubstantial inflammatory immune response (i.e. that is capable ofcausing breast tissue failure) in breast tissue. Subsequent encounterwith human α-lactalbumin, such as that expressed by cells of adeveloping tumor elicits a recall response by the immune system. Therecall response includes, but is not limited to, an increase in theproduction of proinflammatory cytokines such as IFNγ and IL-2, whichpromote a robust immune system attack against the α-lactalbuminexpressing cells. In the instance in which human α-lactalbumin isproduced only by cells of the human breast, the proinflammatory immuneresponse will be breast tissue specific.

In one embodiment, a method of immunizing a human patient against humanα-lactalbumin is disclosed. The method comprises the step ofadministering to the patient an immunogenic composition comprising apolypeptide comprising human α-lactalbumin (SEQ ID NO: 1). In oneaspect, the immunogenic composition comprises a polypeptide thatconsists essentially of human α-lactalbumin.

In one embodiment, a method of activating human T cells capable ofinducing a breast tissue specific inflammatory response in a humanpatient is disclosed. The method comprises the step of contacting the Tcells with a composition comprising isolated human dendritic cellspreviously exposed to a polypeptide comprising human α-lactalbumin (SEQID NO: 1). The activated T cells exhibit a recall response whensubsequently presented with human α-lactalbumin. The recall responseincludes the production of proinflammatory cytokines and or chemokines,including, for example, IFNγ.

In one embodiment, a vaccine for preventing or treating breast cancer isdisclosed. The vaccine comprises an immunogenic polypeptide comprisinghuman α-lactalbumin. After administration to patients that have breasttissue producing α-lactalbumin, the vaccine induces a breast tissuespecific proinflammatory immune response.

In one embodiment, a method of treating cancer in a human patient isdisclosed. The method comprises the step of administering to the patienta composition comprising human α-lactalbumin, an adjuvant, and apharmaceutically acceptable carrier, in an amount effective to induce abreast tissue specific inflammatory response in the human patient. Inone embodiment, the adjuvant is GPI-0100.

In one embodiment, a method of treating cancer in a human patient isdisclosed. The method comprises the step of administering to the patienta composition, the composition comprising isolated human dendritic cellsthat have been loaded with human α-lactalbumin, in an amount effectiveto induce a breast tissue specific inflammatory response in the humanpatient.

In one embodiment, a method of inducing a breast tissue specificinflammatory response in a human patient is disclosed. The methodcomprises administering to the patient a composition, the compositioncomprising human α-lactalbumin, an adjuvant, and a pharmaceuticallyacceptable carrier, wherein an increase in α-lactalbumin reactive IFNγproducing T cells is produced after administration of the composition.

In one embodiment, a method of inducing a breast tissue specificinflammatory response in a human patient is disclosed. The methodcomprises administering to the patient a composition, the compositioncomprising isolated human dendritic cells that have been loaded withhuman α-lactabumin, wherein an increase in α-lactalbumin reactive IFNγproducing T cells is produced after administration of the composition.

An effective amount of human α-lactalbumin refers to an amount of humanα-lactalbumin that is sufficient to be taken up by antigen presentingcells and/or activate T cells to elicit an immune response.

According to various embodiments for treatment or prevention of breastcancer, one or more booster injections of the vaccine are administered.

T cells recognize discrete peptides of protein antigens presented in thecontext of antigen presenting molecules that are typically expressed onmacrophages and dendritic cells of the immune system. Peptiderecognition typically occurs following phagocytic processing of theantigen by antigen-presenting cells and loading of small peptidefragments onto Major Histocompatibility Complex (MHC) class I and/orclass II molecules. After CD4⁺ T cells recognize peptides presented onMHC class II molecules, they proliferate rapidly and become effector Tcells that may activate other immune effector cells.

CD8⁺ T cells are believed to recognize peptides presented by MHC class Imolecules, upon which they develop into cytotoxic effector cells capableof lysing and eliminating cells that express a particular protein. CD4and CD8 molecules serve as co-receptors because their interactions withMHC molecules. They are believed to be required for an effective T cellmediated immune response.

The ability of human α-lactalbumin to be an effective polypeptideantigen in a vaccine against breast cancer depends on whether humanα-lactalbumin is sufficiently immunogenic in humans to generate aproinflammatory immune response. The immunogenicity of a particularprotein, such as human α-lactalbumin, is highly unpredictable, anddepends in part upon the particular amino acid sequence of the protein,its uptake and processing by antigen presenting cells into smallerpeptide fragments, the availability of appropriate MHC binding sites forthe processed peptide fragments, and the availability of appropriatelyresponsive T cells with specific receptor sequences that can recognizeand bind the peptide in the context of the MHC binding pocket.

EXAMPLES Example 1 α-Lactalbumin Immunization Activates both CD4+ andCD8+Proinflammatory T Cells

Recombinant mouse α-lactalbumin is purified under denaturing conditionsusing nickel-nitrilotriacetic acid affinity chromatography followed byreverse phase HPLC. Female SWXJ mice are immunized with recombinantmouse α-lactalbumin. Ten days after immunization, lymph node cells (LNC)in the mice show a dose-dependent proliferation in recall responses toα-lactalbumin and are unresponsive to recombinant human cochlingenerated in E. coli in a virtually identical manner (see FIG. 1A). BothCD4⁺ and CD8⁺ T cells are involved in responsiveness to α-lactalbumin(see FIG. 1B). Furthermore, α-lactalbumin shows a proinflammatoryphenotype involving a high production of interferon-gamma (IFNγ) andIL-2 and a low production of IL-4, IL-5, and IL-10 (see FIG. 1C).

Example 2 Immunization of Non-Lactating Mice with α-Lactalbumin Fails toInduce Breast Inflammation

Breast tissue from non-lactating mice immunized with α-lactalbumin doesnot demonstrate inflammatory infiltration, but instead consistentlyshows isolated individual CD3⁺ T cells migrating through breastparenchyma. However, extensive T cell infiltrates consistently occurthroughout the mammary tissue of lactating mice immunized withα-lactalbumin. Breast tissue from lactating control mice immunized withCFA alone does not show inflammatory T cell infiltration. Analysis ofbreast infiltrating T cells by flow cytometry shows a high frequency ofCD3⁺CD4⁺ T cells and CD3⁺CD8⁺ T cells expressing the CD44^(high)activation marker. Analysis by quantitative real-time RT-PCR shows thatbreast tissue from lactating mice immunized with α-lactalbumin havesignificantly elevated expression levels of IFNγ (p=0.001) but not IL-10(p>0.10) compared to levels expressed in breast tissue from untreatednormal non-lactating or lactating mice, or from lactating mice immunizedwith CFA alone (see FIG. 2).

Example 3 Prophylactic α-Lactalbumin Vaccination Inhibits Growth ofBreast Tumors

MMTV-neu mice express the unactivated neu (ErbB2 or HER2/neu)protooncogene under the regulation of the long terminal repeat of mousemammary tumor virus (MMTV) and show a 50% incidence of spontaneousmammary tumors by 205 days of age. Eight week old MMTV-neu mice areimmunized with either α-lactalbumin in CFA or with CFA alone. All miceare euthanized when the first tumor reached 17 mm in diameter (at around10 months of age). Upon completion of the experiment, all CFA-immunizedcontrol mice develop breast tumors upon. In comparison, none of the miceimmunized with α-lactalbumin show any detectable mammary tumors(p=0.0004; see FIG. 3A).

Prophylactic vaccination with α-lactalbumin is also effective againsttransplantable 4T1 tumors. BALB/c mice immunized with α-lactalbumin 13days prior to inoculation with 4T1 tumor cells exhibit significantgrowth inhibition (p=0.0006; see FIG. 3B).

Example 4 α-Lactalbumin Vaccination Inhibits Growth of EstablishedTransplanted 4T1 Breast Tumors

Following subcutaneous inoculation of BALB/c mice with 2×10⁴ 4T1 tumorcells, tumors are well established within 5 days after inoculation andpalpable tumors are present within 2 to 3 weeks after inoculation. Afterinoculation with 4T1 tumor cells, vaccination with α-lactalbumin isperformed at 5 days after inoculation, at 13 days after inoculation, andat 21 days after inoculation. A significant inhibition of tumor growthis observed at the 5-day vaccination (p<0.01; see FIG. 4A) and at the13-day vaccination (p<0.01; see FIG. 4B) but not at the 21-dayvaccination (see FIG. 4C). The lack of tumor growth inhibition in micevaccinated 21 days after inoculation may be due to the shortened 11-dayobservation period between the time of immunization and the time whentumors reach the maximum size mandating euthanasia.

Example 5 α-Lactalbumin Vaccination Inhibits Growth of EstablishedAutochthonous Breast Tumors

MMTV-PyVT transgenic mice demonstrate loss of lactational abilitycoincident with transgene expression and develop palpable veryaggressively growing mammary tumors by 5 weeks of age. In this example,MMTV-PyVT transgenic mice are vaccinated at 6 weeks of age withα-lactalbumin. Significant inhibition in the growth of very aggressiveestablished autochthonous tumors in MMTV-PyVT is observed (p<0.0006; seeFIG. 5). Thus, α-lactalbumin vaccination indicates effective protectionand therapy against breast tumor growth and is particularly effectivewhen immunization occurs prior to the appearance of palpable tumors inMMTV-PyVT transgenic mice.

Example 6 α-Lactalbumin-Specific T Cells Induce Tumor Inflammation andCytotoxicity

BALB/c mice are vaccinated with α-lactalbumin and inoculated with 4T1cells. Approximately 32 days after inoculation, tumors in the BALB/cmice show extensive infiltration of CD3⁺ T cells. In comparison, theseinflammatory infiltrates do not occur in tumors from control miceimmunized with CFA. How cytometry analysis of tumor infiltratinglymphocytes (TILs) show a predominance of CD4⁺ (64.3%) T cells comparedto CD8⁺ (14.4%) T cells.

Furthermore, recall responses to 50 μg/ml α-lactalbumin as measured byELISA demonstrate a type-1 proinflammatory phenotype involving highproduction of IFNγ compared to IL-5 and IL-10 (see FIG. 6A) ELISPOTanalysis of TILs shows that CD4⁺ rather than CD8⁺ T cells are producedthe IFNγ since its secretion by cultured T cells is inhibited bytreatment with class II but not class I specific antibodies (see FIG.6B). However, death of cultured 4T1 tumor cells is inhibited bytreatment of cultured α-lactalbumin primed LNC with antibodies specificfor mouse CD8 but not CD4 (see FIG. 6C). This result indicates that CD8⁺T cells mediate 4T1 specific cytotoxicity.

Example 7 Inhibition of Breast Tumor Growth by α-Lactalbumin Vaccinationis Mediated by T Cells

On the same day, naïve recipient BALB/c mice are inoculated with 4T1tumors and α-lactalbumin-primed LNC. A significant inhibition of tumorgrowth is observed in these mice (p<0.0001; see FIG. 7A). Furthermore,the incidence of tumor bearing mice is significantly decreased in thisexample (p<0.03; see FIG. 7B) and the final tumor weight is alsosignificantly decreased (p<0.0008; see FIG. 7C).

Naïve mice further received a) CD4⁺ T cells enriched by magnetic beadseparation from α-lactalbumin-primed LNC, b) CD8⁺ T cells enriched bymagnetic bead separation from α-lactalbumin-primed LNC, or c) controlovalbumin (OVA)-primed LNC. Significant tumor growth inhibition isobserved in the mice receiving the CD4⁺ T cells enriched by magneticbead separation from α-lactalbumin-primed LNC (p=0.002; see FIG. 7D,left panel) and the CD8⁺ T cells enriched by magnetic bead separationfrom α-lactalbumin-primed LNC (p=0.003; see FIG. 7D, right panel)compared to OVA-primed LNC. This example indicates that activated CD4⁺and CD8⁺ TILs mediate the protective and therapeutic effects ofα-lactalbumin vaccination on breast tumor growth.

Example 8 Availability of α-Lactalbumin Responsive T Cells in Females

T cell repertoire availability and magnitude is assessed in peripheralblood mononuclear cells (PBMC) by in vitro priming against α-lactalbuminand measurement of the resulting antigen-specific frequencies ofIFNγ-producing T cells. Monocyte derived DCs were prepared from PBMCtaken from a 29 year-old female patient. Adherent cell selection wasfollowed by culture in X-VIVO media (BioWhittaker, Walkersville, Md.)with 500 U/ml rhGMCSF and rhlL-4 (Peprotech, Rocky Hill, N.J.). Six daysafter initiation of culture, DCs were pulsed with 75 μg/ml of purifiedrecombinant human α-lactalbumin (rhα-lactalbumin) and were washedextensively 48 hours later. The washed DCs were co-cultured with nylonwool purified naive T cells from the same donor at a ratio of 1:5 (DCsto T cells). Approximately 72 hours after co-culture, in vitro primed Tcells and unprimed T cells from the same donor were enriched by passagethrough nylon wool and re-cultured with γ-irradiated (3000 rads) PBMC asfeeder cells at a ratio of 1:10 (feeders to T cells) on ELISPOT plates(Polyfiltronics, Rockland, Mass.) pre-coated with mouse anti-human IFNγcapture antibody (#M-700A; Endogen, Cambridge, Mass.). Frequencies ofα-lactalbumin reactive IFNγ producing T cells were determined 48 hourslater using secondary biotinylated mouse anti-human IFNγ (#M701;Endogen) and resolution of ELISPOTS using an automated ImmunospotSatellite Analyzer (Cellular Technology, Cleveland, Ohio).

After priming with α-lactalbumin, the PMBCs demonstrate an increasedfrequency of IFNγ producing T cells (see FIG. 8) upon subsequentexposure to α-lactalbumin (recall response). The observed response isantigen specific, as recall antigens OVA and recombinant human cochlin(rmCochlin), an inner ear protein generated in transduced E. coli in amanner similar to the production of recombinant human α-lactalbumin, donot elicit an increase in IFNγ producing T cells.

Taken together, the results described herein show that show that 1)immunization with α-lactalbumin activates both CD4⁺ and CD8⁺proinflammatory T cells; 2) immunization of non-lactating mammals withα-lactalbumin fails to induce breast inflammation; 3) prophylacticα-lactalbumin vaccination inhibits growth and incidence of breasttumors; and 4) α-lactalbumin vaccination inhibits growth of establishedtumors. α-lactalbumin immunization provides a safe and effectivevaccination in several murine breast cancer models.

Importantly, it is also demonstrated herein that human α-lactalbumin issufficiently immunogenic in humans to activate T cells and elicit aproinflammatory immune recall response. Thus, immunization of humanswith human α-lactalbumin has the ability to provide a safe and effectivevaccine for human breast cancer.

While the invention has been illustrated and described in detail in theforegoing description, such an illustration and description is to beconsidered as exemplary and not restrictive in character, it beingunderstood that only the illustrative embodiments have been describedand that all changes and modifications that come within the scope of theinvention are desired to be protected. Those of ordinary skill in theart may readily devise their own implementations that incorporate one ormore of the features described herein, and thus fall within the scope ofthe present invention.

What is claimed is:
 1. A method of inducing production of IFNγ bypurified human female CD4+ or CD8+ T cells, said method comprising thestep of exposing purified human female antigen presenting cells to anantigen consisting of recombinant human α-lactalbumin as set forth inSEQ ID NO: 1 in the presence of purified human female CD4+ or CD8+ Tcells, wherein said exposing step activates said purified human femaleCD4+ or CD8+ T cells, thereby inducing production of IFNγ by thepurified human female CD4+ or CD8+ T cells.
 2. The method of claim 1,wherein the antigen presenting cells comprise dendritic cells.
 3. Themethod of claim 2, wherein the dendritic cells are prepared fromperipheral blood mononuclear cells isolated from a human female.
 4. Themethod of claim 1, wherein the human female CD4+ or CD8+T cells comprisepurified unprimed CD4+ or CD8+T cells from a human female.
 5. The methodof claim 4, wherein the human female CD4+ or CD8+T cells comprise CD4+Tcells.
 6. The method of claim 4, wherein the human CD4+ or CD8+T cellscomprise CD8+ T cells.
 7. The method of claim 1, wherein the recombinantα-lactalbumin is produced in Escherichia coli.